N'-(3'-Monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine (1A) is a biomarker of benzidine exposure, an endproduct of initiation, and produces genotoxic lesions. Prostaglandin H synthase (PHS) activates the aromatic amine bladder carcinogen N-acetylbenzidine (ABZ) to form 1A. This report examines the mechanism of PHS metabolism of ABZ. Arachidonic acid (0.2 mM) with ram seminal vesicle microsomes convert 3H-ABZ (0.06 mM) to 4'-nitro-4-acetylaminobiphenyl (21% of total metabolism by HPLC). With ascorbic acid, N'-hydroxy-N-acetylbenzidine (N'HA) was the major product and was identified by ESI/MS/MS. Similar results were observed with H2O2 (0.3 mM). Cyanide (10 mM), a peroxidase inhibitor, prevented metabolism, but cytochrome P-450 inhibitors SKF-525A, furafylline, or (-naphtho-flavone (0.1 mM) did not. The lack of effect of DMPO suggests that radical products generated by electron transfer were not involved. With H2O2, oxygen uptake was not detected. Neither h ydroxyl ra dicals nor superoxide are sources of oxygen incorporation, because mannitol and superoxide dismutase did not inhibit. To determine the source of oxygen incorporated into ABZ, [18O]H2O2 was used. 18O-N'HA enrichment was compared to [18O]H2O2 alkaline oxidation of menadione to its 18O-epoxide. N'HA enrichment was similar to the epoxide. Thus, PHS metabolizes ABZ by peroxygenation. N'HA may form 1A during PHS metabolism of ABZ.